Eukaryotic cells are characterized by numerous membrane-bounded compartments that perform specialized functions essential for the cell. Protein targeting and translocation systems maintain the proper composition of these compartments. Mis-targeting of proteins can lead to disease. Examples of this are I-cell and hyperoxaluria I, where enzymes are localized to the wrong compartment. The long range goal of our research is to understand the molecular basis and biochemical mechanisms of a variety of targeting and translocation systems. The investigator's are particularly interested in the evolution of translocation systems to accommodate assembly requirements of their substrates. Eukaryotic cells consist of protein import systems and protein export systems. The latter derive from systems present in the bacterial cell membrane. During the last two years, two new export systems have been described. One, called the Delta pH system, was originally discovered in the thylakoied membrane of chloroplasts, but appears to be widely present in prokaryotes and also in eukaryotic organelles. The Delta pH system specifically transports a subset of proteins to the lumen in parallel with the well-studied SecA/SecY system. The identified components of the Delta pH system are unlike those of the Sec system. The proposed work aims to determine the basis for the existence of these two parallel systems and to elucidate the mechanisms by which the Delta pH system recognizes and transports it proteins. The first specific aim is to determine if the Delta pH system can transport folded proteins and if substrates of the Delta pH system need to be folded during transport. The second specific aim is to investigate the mechanism of the Delta pH system by characterizing two intermediates of the pathway. Finally, they propose experiments to develop in vitro and in vivo assays that will examine the functions and the functional motifs of components of the Delta pH system. Genetic studies have revealed the in vivo necessity of multiple protein transport systems in a number of cell membranes. Therefore, results of the proposed studies will be generally applicable and relevant to basic cell biology and human health.